Enteroviruses (EVs) 3C proteins suppress type Ⅰ interferon (IFN) responses mediated by retinoid acid-inducible gene Ⅰ(RIG-I),while an E3 ubiquitin ligase,tripartite motif protein 25 (TRIM25)-mediated RIG-I ubiquitination is essential for RIG-I antiviral activity.Therefore,whether the effect of EVs 3C on RIG-I is associated with TRIM25 expression is worth to be further investigated.Here,we demonstrate that 3C proteins of EV71 and coxsackievirus B3 (CVB3) reduced not only RIG-I expression but also TRIM25 expression through protease cleavage activity,while overexpression of TRIM25 restored RIG-I expression and IFN-β production reduced by 3C proteins.Further investigation confirmed that the two amino acids and functional domains in TRIM25 required for RIG-I ubiquitination and TRIM25 structural conformation were essential for the recovery of RIG-I expression.Moreover,we also observed that TRIM25 could rescue RIG-I expression reduced by 3C proteins of CVA6 and EV-D68 but not CVA16· Our findings provide an insightful interpretation of 3C-mediated host innate immune suppression and support TRIM25 as an attractive target against multiple EVs infection.